Field of the Invention
This invention relates to anticancer agents and the processes for their preparation.
The process of the invention for the preparation of N-{[5-(2,4-diamino-6-pteridinyl)ethyl]-2-theonyl}-L-glutamic acid (1) is illustrated in Scheme I and explained by Example 1., where the compound number identify the same compounds which they identify in all descriptions.
Cancer is a disease that is characterized by abnormal tissue growth and destruction and this acute or chronic disease of humans can be treated effectively with antifolate drugs such as methotrexate (MTX).
Methotrexate is a potent inhibitor of the enzyme dihydrofolate reductase, and thereby, it depletes the cells of various forms of the vitamin folic acid, that are required for cell division. Baugh, Krumdieck and Nair reported [Biochemical and Biophysical Research Communications 52:27 (1973)] that methotrexate is metabolized to its poly-y-glutamates in human tissues, and Nair and Baugh (Biochemistry 12, 3923, 1973) synthesized these metabolites and identified their presence in rodent tissues. Balinska, Galivan and Coward reported in Cancer Research that methotrexate poly-y-glutamates with higher chain lengths are retained longer within the cells (Cancer Res. 41:2751, 1981). Investigations of the biochemical pharmacology of methotrexate polyglutamates revealed beyond doubt that these metabolites are important determinants of anticancer activity and host toxicity. [M. G. Nair, in "Cancer Growth and Progression-Cancer Control in Man", Chapter 10, H. E. Kaiser (Ed.) Kluwer Academic Publishers, 1990); M. G. Nair, In "Chemistry of Antitumor Agents". D. E. V. Wilman (Ed), Blackie and Sons (Lond); Chapman and Hall (USA) Chapter 7 (1990)] Recently, several attempts have been made to reduce the host toxicity of methotrexate and other antifolates by modulating their glutamylation. (M. G. Nair, and Ann Abraham, U.S. Pat. No. 4,996,207 (1991); M. G. Nair, U.S. Pat. No. 5,073,554). In addition to the utility of classical antifolates as anticancer agents, they are also useful in the treatment of autoimmune diseases such as rheumatoid arthritis. Indeed, a close analogue of methotrexate (Rheumatrex), 10-deazaaminopterin (10-DAM) has been shown to be equally effective as an arthritis remittive drug for the treatment of rheumatoid arthritis in humans (C. L. Krumdieck, O. Castaneda, G. Alarcon, W. J. Koopman and M. G. Nair, U.S. Pat. No. 5,030,634) in a clinical trial. In this context, it was of interest to develop powerful inhibitors of dihydrofolate reductase with altered ability of polyglutamylation, and enhanced tissue penetration as better therapeutic agents for the treatment of neoplastic, autoimmune and inflammatory diseases. Substitution of the benzene ring of 10-deazaaminopterin [J. I. DeGraw, R. L. Kisliuk, Y. Gaumont, C. M. Baugh, and M. G. Nair, J. Med. Chem. 17, 552, (1973); M. G. Nair, J. Org. Chem. 50, 1879 (1985)] with a thiophene ring gave a very potent inhibitor of dihydrofolate reductase, that has demonstrated diminished polyglutamylation and enhanced transport to tumor cells as expected. This new thiophene substituted antifolate (1) and its close derivatives are envisioned to be anticancer and anti-inflammatory drugs exhibiting lower toxicity and enhanced specificity compared to methotrexate (MTX) and 10-deazaaminopterin (10-DAM). Although compound 1 is polyglutamylated less efficiently compared to methotrexate, it exhibits superior activity against the growth of (I.sub.50 10.2 vs 13.5 nM) human leukemia cells due to enhanced transport. Taken together, these new and unexpected results establish that compound 1 and its close analogues should have clinical utility as novel anticancer drugs capable of exhibiting lower host toxicity.
This invention accordingly provides a process for treating leukemia, ascitic and solid tumors and by analogy with methotrexate and 10-deazaaminopterin a process for treating auto-immune diseases such as rheumatoid arthritis and inflammatory diseases such as asthma, which comprises administering to a warm blooded animal with an abnormal proportion of leukocytes or other evidence of malignancy, rheumatoid arthritis, or asthma, a therapeutic nontoxic amount of N-{]5-(2,4-diamino-6-pteridinyl)ethyl]-2-theonyl}-L-glutamic acid (1) as such, or in the form of a pharmacologically acceptable salt thereof.
The salts of 1 may be formed with one or more of the amino groups of the pteridine ring with acids such as hydrochloric, hydrobromic, sulfuric, sulfonic, nitric, phosphoric, or organic carboxylic acids such as acetic, citric, salicylic, or methene sulfonic.
Compound 1 or salts thereof may be administered to a warm blooded animal by oral or parenteral (intraperitoneal, intravenous, intrathecal, subcutaneous, intramuscular, etc.) routes. Higher dosage of 1 may be administered in conjunction with racemic leucovorin [6-(R,S)5-formyltetrahydrofolate] or folic acid to further reduce toxicity in the treatment of cancer and auto-immune diseases such as rheumatoid arthritis.
The new thiophene substituted antifolate 1 may be provided in composite forms to facilitate administration to patients or in dosage unit form. A non-toxic and sterile carrier may be added to 1. This carrier may be a solid, liquid or semi-solid that may serve as a medium, vehicle or excipient. Methyl cellulose, polyhydroxybenzoate, talc, gelatin, lactose, dextrose, starch, mannitol, sorbitol, mineral oil, gum acacia, oil of theobroma or magnesium stearate may serve as the carriers. Compound 1 and carrier or diluent can be encapsulated, or enclosed in a paper or other container, cachet, gelatin, capsule or sachet when intended for use in dosage units.
The process of the invention for the preparation of 1 is a synthesis in which commercially available 2-thiophene carboxaldehyde is converted to the corresponding acetal derivative by reactions with a trialkyl orthoformate using standard procedures. The resulting 2-thiophene dialkyl acetal 5 is converted to a 2-carboxy-5-formylthiophene dialkyl acetal 6 by reacting with a strong base like butyllithium, lithium diisopropylamide (LDA) or potassium-t-butoxide followed by CO.sub.2 in tetrahydrofuran at a temperature range of -60.degree. to 30.degree. C. Esterification of the carboxyl group of 6 to an ester followed by deprotection of the aldehyde gives the ester product, of 5-formyl thiophene-2-carboxyic acid 7. The above conversion of 6 to 7 can be accomplished by treating 6 with thionyl chloride in an appropriate alcohol for 12-24 hours and then stirring the reaction mixture with H.sub.2 O for 3 hours. 1-Phthalimido-3-(triphenylphosphoranylidine)-2-propanone(synthesized according to the literature procedure of (Nair, M. G., J. Org. Chem. 50, 1879, 1985) is refluxed in CH.sub.2 Cl.sub.2 with 7 to prepare 4-(2-carbomethoxytheonyl)-1-pthalimido-3-buten-2-one (8). The enone 8 can be reduced to 1-phthalimido-4-(2-carbomethoxytheonyl)-2-butanone (9) by refluxing with palladium/carbon in formic acid/triethylamine mixture for 18 hr. This reaction can also be accomplished with Zn/CH.sub.3 COOH or by catalytic hydrogenation using catalysts such as Palladium or Platinum on carbon.
The ketone product (9) was protected as the oxime 1-Phthalimido-4-[(2-carbomethoxy)-5-theonyl]-2-butanone oxime (10) by standard procedure. The oxime was a mixture of syn and anti isomers. The isomeric mixture of the oxime was then subjected to hydrazinolysis following the literature procedure of Nair et al, (J. Org. Chem. 40, 1745, 1975) to obtain 1-amino-4-[(2-carbomethoxy)5-theonyl]-2-butanone oxime (11). Compound 11 was then reacted with 6-chloro-2,4-diamino-5-nitropyrimidine to obtain 1-[(2,4-diamino-5-nitropyrimidin-6-yl)amino]4-[(2carbomethoxy)-5-theonyl]- 2-butanone oxime (12).
The next stage of synthesis is the conversion of 12 to 13, a procedure that involves the deprotection of the oxime mixture with a solution of TFA and 1N HCl, followed by reduction of the nitro group to the amino group by sodium dithionite in aqueous DMF. The resulting reduction product could then be cyclized with the use of inorganic aqueous base and oxidized with 5% KMnO.sub.4 or by heating the reduction product in a solution of DMF to 100.degree. C. for 1 h and subsequent hydrolysis to obtain 14.
The final stage of the synthesis is the coupling of a diester of L-glutamic acid such as diethyl-L-glutamate or di-t-butylglutamate to 14, first by converting 14 to the corresponding mixed anhydride with a suitable alkylchloroformate in THF or DMF in presence of a tertiary organic base and then reacting the resulting mixed anhdyride with the L-glutamate diester and subsequent workup. The product is then treated with a solution of inorganic base like sodium hydroxide or potassium hydroxide to obtain the sodium or porassium salt of compound 1. Acidification of the hydrolysate with acetic acid gives a precipate of 1, which can be filtered and purified further.
Compound 1 is a potent inhibitor of L1210 dihydrofolate reductase (DHFR). DHFR inhibitory data for 1 and MTX under identical experimental conditions are shown in Table I.
TABLE I ______________________________________ Inhibition of DHFR by 1 and MTX COMPOUND I.sub.50 (nM) ______________________________________ 1 0.95 MTX 0.98 ______________________________________
Compound 1 showed excellent inhibitory activity against the growth of CCRF-CEM human leukemia cells, and H35 hepatoma cells. (Table II). CCRF-CEM human leukemia cells (Table II) were more sensitive to 1 than methotrexate.
TABLE II ______________________________________ Inhibition of tumor cell growth by 1 and methotrexate I.sub.50 (nM) CCRF-CEM Compound Human leukemia H35 hepatoma ______________________________________ 1 10.2 15 Methotrexate 13.5 10 ______________________________________
Growth inhibition of H35 hepatoma cells was measured as described by Patil, Jones, Nair, Galivan, Maley, Kisliuk, Gaumont, Duch, and Ferone in the Journal of Medicinal Chemistry 32:1284 (1989) and that of CCRF-CEM human leukemia cells according to the procedures of McGuire, Graber, Licato, Vincenz, Coward, Nimec and Galivan (Cancer Research 49:4517, 1989).
The substrate activity of compound 1 as shown in Table III established that it is polyglutamylated. It has been shown previously that substrates of FPGS are capable of polyglutamylation in vivo and the relative magnitude of substrate activity of an antifolate to this enzyme compared to a standard is a measure of its relative ability to undergo polyglutamylation. The v/k values of Table III established that compound 1 is polyglutamylated less efficiently than either aminopterin or 10-deazaaminopterin.
TABLE III ______________________________________ Substrate activity with CCRG-CEM human luekemia cell FPGS* COMPOUND K.sub.m (.mu.M) Vmax V/K ______________________________________ Aminopterin 4.6 100 21.7 10-DAM 34.8 88.6 2.5 1 45.0 83.5 1.9 ______________________________________ *Assay as described by McGuire, Bolanowska, and Piper in Biochem. Pharmacol 37; 3931, 1988.
Transport studies showed that compound 1 is transported more efficiently to H35 hepatoma cells in culture than methotrexate (TABLE IV). Transport influx was estimated by measuring the ability of 1 to complete with folinic acid transport to this cell line.
TABLE IV ______________________________________ Inhibition of folinic acid transport to H35 hepatoma cells. COMPOUND I.sub.50 (.mu.M) ______________________________________ 1 5 Methotrexate 18 ______________________________________
Transport experiments were conducted as described by Patil, Jones, Nair, Galivan, Maley, Kisliuk, Gaumont, Duch and Ferone in the Journal of Medicinal Chemistry 32: 1284 (1989). Folinic acid 2 .mu.M, 15 min, uptake.